Drug repurposing: insights into the antimicrobial effects of AKBA against MRSA

Staphylococcus aureus is a major threat in infectious diseases due to its varied infection types and increased resistance. S. aureus could form persister cells under certain condition and could also attach on medical apparatus to form biofilms, which exhibited extremely high resistance to antibiotics. 3-Acetyl-11-keto-beta-boswellic acid (AKBA) is a well-studied anti-tumor and antioxidant drug. This study is aimed to determine the antimicrobial effects of AKBA against S. aureus and its persister cells and biofilms. The in vitro antimicrobial susceptibility of AKBA was assessed by micro-dilution assay, disc diffusion assay and time-killing assay. Drug combination between AKBA and conventional antibiotics was detected by checkerboard assay. And the antibiofilm effects of AKBA against S. aureus were explored by crystal violet staining combined with SYTO/PI probes staining. Next, RBC lysis activity and CCK-8 kit were used to determine the cytotoxicity of AKBA. In addition, murine subcutaneous abscess model was used to assess the antimicrobial effects of AKBA in vivo. Our results revealed that AKBA was found to show effective antimicrobial activity against methicillin-resistant S. aureus (MRSA) with the minimal inhibitory concentration of 4–8 µg/mL with undetectable cytotoxicity. And no resistant mutation was induced by AKBA after 20 days of consecutive passage. Further, we found that AKBA could be synergy with gentamycin or amikacin against S. aureus and its clinical isolates. By crystal violet and SYTO9/PI staining, AKBA exhibited strong biofilm inhibitory and eradication effects at the concentration of 1 ~ 4 µg/mL. In addition, the effective antimicrobial effect was verified in vivo in a mouse model. And no detectable in vivo toxicity was found. These results indicated that AKBA has great potential to development as an alternative treatment for the refractory S. aureus infections. Supplementary Information The online version contains supplementary material available at 10.1186/s13568-024-01660-0.


Introduction
Staphylococcus aureus (S. aureus) is a common Grampositive pathogen that often causes skin and soft tissue infections, respiratory infections, osteomyelitis, and medical device implant related infections in patients with immunodeficiency.In severe cases, it can lead to lethal blood infections such as sepsis (Vestergaard et al. 2019;2014;Chang et al. 2020).Due to the non-proliferation and inactive metabolism of the persister cells, as well as the inhibition of ATP production, their drug resistance is greatly increased.Persister cells formed by S. aureus are resistant to almost all commoditized antibiotics, and are one of the main reasons for the persistence of refractory infections (Chang et al. 2020).Therefore, developing new antibacterials targeting MRSA and its persister cells has become an urgent problem to be solved.
S. aureus can adhere to the surface of medical implants and further aggregate to form a biofilm (Lister and Horswill 2014).The extracellular matrix of the biofilm can effectively prevent the penetration of antibiotics, thereby greatly increased the bacterial resistance.The use of conventional antibiotics alone often makes it difficult to effectively eradicate the biofilm that has already preformed, posing great difficulties for clinical treatment (Guo et al. 2022).Therefore, studying antibacterial drugs targeting mutidrug-resistant S. aureus and its biofilms is of great clinical significance.
Drug repurposing, which refers to the reuse of wellstudied drugs apart from its original targets, has been a research hotspot in recent years.It has many irreplaceable advantages over traditional drug development.Most of these drugs have clear targets, pharmacokinetic and toxicological data, which greatly save research time and costs (Zheng et al. 2018).And drug repurposing is particularly suitable for the development of drugs for explosive epidemics.For example, in recent years, by drug repurposing, researchers found that the well marketed drugs, such as radcivir, favepiravir, ribavirin, lobinavir, litonavir, darunavir, abido, chloroquine and hydroxychloroquine, have significant antiviral effects against coronavirus (Singh et al. 2020).
3-Acetyl-11-keto-beta-boswellic acid (AKBA), a pentacyclic triterpenoid, is a well-studied anti-tumor as well as antioxidant reagent (Ding et al. 2016;Kumar et al. 2023).And it also shows biological activities in nervous system diseases (Gong et al. 2022).However, to the best of our knowledge, there is no study reported the antimicrobial effects of AKBA against S. aureus.Thus, in our present study, we will explore the in vitro and in vivo antimicrobial activities of AKBA, and further assess its effects against S. aureus high resistant phenotypes, for example, biofilms and persister cells.

Bacterial strains and culture conditions
The MRSA strains (including ATCC 43,300 and USA300), MSSA strains (including ATCC 25,923 and MW2) (Tiandiren Biotech, Changsha, China).Clinical strains of MRSA were isolated from the Third Xiangya Hospital of Central South University, and identified by Matrix-Assisted Laser Desorption Ionization (BD, Germany).S. aureus and S. epidermidis were grown in Tryptic Soy Broth (TSB), E. faecalis was grown in Brain Heart Infusion (BHI).Other Gram-negative strains were grown in Luria-Bertani (LB) broth.All the culture mediums were purchased form Solarbio (Beijing, China).Chemicals of AKBA and other antibiotics were purchased from the MedChem Express (New Jersey, USA) and dissolved in dimethyl sulfoxide (DMSO) or deionized water.

Micro-dilution assay for antimicrobial susceptibility determination
The antimicrobial susceptibility of antimicrobials against pathogens was determined according to the recommendation of the Clinical and Laboratory Standards Institute (CLSI) (CLSI 2023).Briefly, single colony of overnight cultured S. aureus was adjusted to 0.5 McFarland (McF) in Mueller-Hinton (MH) II broth.Then, the bacterial suspension was 1: 100 diluted with MH borth, and mixed with equal volumes of 2-fold diluted antimicrobials.After incubation at 37℃ for 16-18 h, the minimal inhibitory concentration (MIC) was determined as the lowest concentration with no visible bacterial growth.Further, an aliquot of supernatant form 1× MIC to the uppermost concentration was removed on a MH agar.After overnight incubation, the minimal bactericidal concentration (MBC) was determined as the lowest concentration with no visible colony formation.

Disc diffusion assay
The procedure was designed in accordance with the recommendation of the CLSI (CLSI 2023).The log-phased S. aureus ATCC 43,300 was adjusted to 0.5 McF with saline, and the bacterial suspension was spread onto the MH agar plate with a sterile swab.The commercial blank disks (diameter = 6 mm) containing DMSO or AKBA were loaded on the surface.After incubating for 16-18 h at 37 ℃, the inhibitory zone was measured by a caliper.

Time-killing assay
S. aureus was cultured to Log-phased growth and adjusted to ~ 1 × 10 6 CFU/mL in MH broth in centrifuge tubes.Equal volumes of 2-fold MH broth diluted antimicrobials were added to each tube.The bacterial suspension was then incubated at 37 ℃ at 180 rpm.An aliquot of the suspension was removed to sheep blood agar to perform CFU counting at the time point of 0, 2, 4, 8, 12 and 24 h, respectively (Liu Y. 2021).

Drug combination determination
The antimicrobial interactions between AKBA and conventional antibiotics were evaluated by checkerboard assay (Mahomoodally et al. 2018).Firstly, the MICs of antimicrobials to be tested were determined as described above.Then, 2-fold dilutions of two drugs were mixed horizontally and vertically, respectively, at equal volumes in a 96-well plate in the presence of ~ 1 × 10 6 CFU/mL S. aureus.After incubated at 37 ℃ for 16-18 h, the OD 630nm was obtained, and the calculation formula for the fractional inhibition concentration index (FICI) is as follows:

Biofilm detection by crystal violet staining
Crystal violet staining is a commonly used method for detecting and visualizing biofilms (Liu et al. 2023).
Briefly, for biofilm inhibition, overnight cultured S. aureus was diluted with TSB to a final concentration of ~ 1 × 10 6 CFU/mL.Equal volumes of the bacterial suspension and 2-fold serially diluted AKBA were added in a 96-well cell culture plates.After incubation at 37℃ for 24 h, the plates were washed to remove any loosely attached cells or debris.The left biofilms were fixed with formaldehyde, and then stained with 0.25% (wt/vol) crystal violet for 20 min.Excess crystal violet dye was washed by PBS, and 100 µL of ethanol was added to each well to destained the crystal violet.The absorbance at 570 nm (A 570nm ) was detected with a microplate reader (Bio-Rad, Hercules, USA) to quantify the biofilm biomass.As for biofilm eradication, the overnight cultured S. aureus was 1: 200 diluted with TSB, and added to a 96-well cell culture plates.After 24 h incubation at 37℃ the excessive planktonic cells were removed with PBS, and 200 µL of 2-fold serially diluted AKBA was added to each well.After incubation at 37℃ for 24 h, the plates were washed with PBS, and stained with crystal violet as described above.

Biofilm detection by confocal laser scanning microscopy (CLSM)
The biofilms were cultured and treated with AKBA as described above in glass-bottomed cell culture dishes (NEST, Wuxi, China).After incubation at 37 °C for 24 h, the dishes were washed with PBS to remove unattached cells.Then, the dishes were stained with 500 µL PBS in the presence of 10 µM SYTO-9/PI (Thermo Fisher Scientific, Waltham, MA, USA) in the dark for 30 min.And the stained biofilms were observed by a CLSM (Zeiss LSM 800, Jena, Germany) with excitation/emission wavelengths of 488 nm/550 nm (SYTO9) and 540 nm/620 nm (PI), respectively (Zhang et al. 2023).

Persister cells killing assay
The persister cells were cultured as described by Liu et al. (Liu et al. 2021).Briefly, S. aureus was prepared at 37 °C with shaking at 200 rpm for 24 h to platform stage.
The bacterial suspension was washed three times with PBS, and adjusted to OD630 = 0.2 in centrifuge tubes.
A 1 ~ 8×MIC of AKBA was added into each tube, and CFU counts were performed after incubation at 37℃ for 0, 2, 4, 6 and 8 h, respectively.Conventional antibiotics of VAN,CIP,DAP,and GEN (10×MIC) were used as controls.
Resistance inducing assay S. aureus was overnight cultured at 37℃ 180 rpm in TSB.
And the bacterial suspension was further sub-cultured in fresh TSB to log-phased growth.The MIC was detected as mentioned above at the first day.After incubation at 37℃ for 24 h, the MIC was recorded, and the bacterial suspension in the wells of 0.5×MIC was 1000-fold diluted with MH broth, and further performed the MIC detection for the next day.Ciprofloxacin was used as a control during the test.The S. aureus was treated for a total of 20 days of serial passage in duplicate (Kim et al. 2018).

RBC lysis activity determination
Human RBC was purchased from the Hemo Pharmaceutical and Biological Co (Shanghai, China).After centrifugation at 1000×rpm for 10 min, RBC was separated and further resuspended in PBS.Indicated concentrations of AKBA were added in equal volumes to RBC solution and incubated at 37 °C for 1 h.After centrifugation at 1000×g for 10 min, the absorbance at 540 nm (A 540 ) was measured.Treatments with PBS and 1% TritonX-100 were used as negative and positive control, respectively (Farheen et al. 2023).The hemolysis rate was calculated as follows:

Cytotoxicity detected by CCK-8 kit
The cytotoxicity of AKBA was determined as previously reported by Liu et al. (Liu et al. 2020)  At an hour post-infection, a single dose of the compounds was administered (s.c.).After 24 h post infection, the mice were euthanized, and the abscess area was calculated with calipers.And the infected skin was dissected and homogenized for bacterial load counting, H&E staining, and immunohistochemical (IL-1β, IL-6, and TNF-α), respectively.Meanwhile, to assess the in vivo toxicity by AKBA, the blood samples were collected from the orbital vein, and the level of organic function biomarkers were determined by Hitachi 7600 series automated biochemistry analyzer (Zhang et al. 2021).

Statistical analysis
All experiments were performed independently in triplicate.The data were analyzed using Prism 8.0 (GraphPad Software, San Diego, CA, USA) and were expressed as the mean ± standard deviation (SD).Statistical significance was determined using unpaired Student's t test or one-way ANOVA and Dunn's multiple comparison test.
A P value of < 0.05 was considered statistically significant.

Bactericidal antimicrobial activity of AKBA against MRSA
Firstly, the antimicrobial susceptibility of AKBA against the Gram-positive and Gram-negative pathogens were determined by micro-dilution assay.As shown in Table 1, AKBA exhibited effective antimicrobial activity against S. aureus (including MSSA and MRSA) with the MIC and MBC of 4-8 µg/mL and 8-16 µg/mL, respectively.Meanwhile, the S. epidermidis also showed the similar susceptibility to AKBA.However, no antimicrobial effect was observed by AKBA against Gram-negative pathogens or fungi with the MIC > 32 µg/mL.Further, the antimicrobial effect by AKBA against MRSA was further confirmed by the disc diffusion assay.As shown in Fig. 1A, MRSA ATCC 43,300 was resistant to gentamycin but susceptible to AKBA with the significantly increased inhibition zone (Fig. 1B).By growth inhibition assay, AKBA was found to significantly inhibit the MRSA ATCC 43,300 growth in TSB at the concentration of sub-MIC (4 µg/ mL) (Fig. 1C), similarly, AKBA exhibited fully bactericidal activity against MRSA at the concentration of MIC (Fig. 1D).And the bactericidal effects could be last for 24 h (Fig. 1D and E).
In addition, no resistant mutation was occurred by MRSA ATCC 43,300 after 20 days of consecutive induction by sub-MIC of AKBA, although 16-to 32-fold of MIC increasement was observed by CIP treatment (Fig. 2A).And no cross-resistance was exhibited with the MIC and MBC of AKBA against CIP-induced resistant strains are still 8 µg/mL (Fig. 2B).Similarly, no resistant (Fig. 2C) or cross-resistant (Fig. 2D) by AKBA against MSSA ATCC 25,923 was exhibited.
To intensify the clinical application and reduce the toxicity, drug combinational effects between AKBA and conventional antibiotics were explored.As shown in Fig. 3, AKBA exhibited synergistical antimicrobial activity with gentamycin (GEN) and amikacin (AMK) with the FICI of 0.5 both.However, no interaction was found between AKBA and other antibiotics with all the FICIs > 0.5.Further, we confirmed the synergy between AKBA with GEN (Table 2) or AMK (Table 3) in clinical isolates of MRSA and MSSA, respectively.

Effective antibiofilm and anti-persister cells effects by AKBA
As widely reported, biofilms formed by MRSA showed high resistant to antimicrobials.However, in the present study, AKBA exhibited significant antibiofilm activity against MRSA.The crystal violet staining assay exhibited that AKBA could significantly inhibit the biofilm formation (Fig. 4A) and eradicated the preformed biofilms (Fig. 4B) at the concentration of 1 and 4 µg/mL, respectively, in a dose-dependent manner.The CLSM images by SYTO9/PI staining also confirmed the biofilm inhibitory (Fig. 4C and D) and eradication (Fig. 4E and F) effects by AKBA with reduced total biofilm biomass as well as the live cells in biofilms.Similarly, AKBA also showed effective bactericidal activity against MRSA persister cells at the concentration of 1-8x MIC in a dose-and timedependent manner, although no antimicrobial effect was observed by 10× MIC of conventional antibiotics of VAN, CIP, daptomycin (DAP) and GEN (Fig. 4G).

Low cytotoxicity of AKBA to mammal cells
Firstly, the human RBC hemolysis activity after AKBA treatment was detected.As we expected, AKBA showed no or only weak hemolysis even at the concentration up to 64 µg/mL (Fig. 5A).Further, the cytotoxicity of AKBA against mammal cells was determined.As shown in Fig. 5B and D, no significant cytotoxicity was observed in the cell lines of LO2 (Fig. 5B), HepG2 (Fig. 5C), and HSF (Fig. 5D).In all, AKBA is well-tolerant to human-derived cells with low hemolysis activity.

In vivo antimicrobial activity and toxicity by AKBA
The antimicrobial efficacy of AKBA in vivo was detected in an abscess infection model.AKBA could significantly reduce the abscess area from (90.33 ± 10.39) to (38 ± 8.76) mm 2 (Fig. 6A), and the viable cell counts was also significantly decreased from (6.57 ± 0.29) to (3.44 ± 0.29) Log10 CFU/mL after treatment with AKBA for 24 h, which even exhibited better antimicrobial activity than VAN (Fig. 6B).In consistence, By H&E staining observation, AKBA treatment largely reduced the inflammatory cells infiltration compared with the vehicle group or the VAN treated group.Similarly, the IHC images also showed more obvious reduction of inflammatory cytokines (IL-1β, IL-6 and TNF-α) in the AKBA treated group than the VAN treated group (Fig. 6C).Meanwhile, the in vivo toxicity by AKBA was also assessed.As shown in Figure S1A, the mice were well tolerant to 20-40 mg/kg of AKBA treatment.Thus, 40 mg/ kg of AKBA by intraperitoneal injection was used to further assess the in vivo toxicity to organs.As we expected, the levels of the liver function biomarker glutamicpyruvic transaminase (ALT, Figure S1B), kidney function biomarker urea nitrogen (BUN, Figure S1C), and myocardium function biomarker creatine kinase (CK, Figure S1D) exhibited no statistical difference between the vehicle and AKBA treated group.

Discussion
The increased antibiotic resistance of MRSA and its ability to form biofilms bring great challenges to clinical treatment.New antibacterial development has become an urgent need.The present study found that the Nrf2 activator AKBA showed effective antibacterial activity against MRSA and its biofilms with low probability of resistance reduction.Meanwhile, synergistical antimicrobial activity between AKBA and GEN or AMK was also observed.In vivo toxicity and antimicrobial efficacy were also assessed in an abscess infection model.The strong antibacterial activity and low cytotoxicity of AKBA makes it potentially to be an alternative for the treatment of refractory infections caused by S. aureus.
Drug repurposing has many advantages that cannot be replaced by the development of antibiotics in traditional ways.For example, lower overall development costs, shorter development time, and extremely low toxicity risks (Zheng et al. 2018).In this study, AKBA exhibited antibacterial activity against MRSA with MICs of 4-8 µg/mL.And S. epidermidis also showed the same susceptibility to AKBA.In recent years, researchers have discovered some highly promising antibacterial drugs against S. aureus through drug repurposing.For example, retinol like anti-tumor drugs CD437 and CD1530 exhibit significant anti-S.aureus activity at microgram per milliliter level.However, CD437 and CD1530 showed significant cytotoxicity on liver tumor cell HepG2 at the concentration of 3-5 µg/mL.And when its concentration reaches 20 µg/mL, there is also significant cytotoxicity to normal liver cell LO2 (Kim et al. 2018;Tan et al. 2019).Therefore, as repurposed antimicrobials, AKBA is safer than CD437 and CD1530.In addition, in recent years, antibacterial drugs such as auranofin (Sharma et al. 2021), penfluridol (Liu et al. 2021a), and idarubicin (She et al. 2020), which have been newly discovered through drug repurposing.But these drugs are still stagnated in the experimental stage due to some factors such as cytotoxicity, in vivo metabolic defects, or stability.However, AKBA not only has strong antibacterial activity, but also S. aureus has a strong ability to adapt to various adverse conditions and to develop resistance to almost all antibiotics.The resistance produced by S. aureus usually poses a serious health burden to the host.These drug resistances are often generated through mutations and rearrangements in the bacterial genome, or through horizontal transmission of drug resistant genes (Pulingam et al. 2022).On the other hand, the emergence of antibiotic resistance phenotype may also be due to the combined effects of the genetic background of the strain and certain environmental factors.For example, indirect regulation of drug resistance can be achieved by altering the expression of resistant genes or the specificity of substrate binding (Pope et al. 1998).In addition, certain insertion elements can alter antibiotic resistance by turning on or off the expression of related genes (Fujimura et al. 2008).In clinical practice, the resistance of S. aureus is usually established through multiple steps.In this study, we found that AKBA with sub-MIC did not induce the development of drug resistance in S. aureus even after 20 days of passage.Therefore, AKBA is extremely difficult to induce bacterial resistance, and has the advantages that conventional antibiotics cannot replace.
AKBA exhibited significant antibiofilm effects against S. aureus.The process of biofilm lifecycle mainly includes initial adhesion, initial biofilm formation, maturation, and dispersion (Liu et al. 2020).The formation of S. aureus biofilm is mainly regulated by the agr-related quorum sensing system.The quorum sensing system can generate self-induced signal peptides.As bacteria gathered, the concentration of the signaling molecules increases, and when exceeded the threshold, the expression of biofilm related genes (such as icaA/B/C/D operon) can be induced.Which will promote the production of extracellular polysaccharides and other biofilm matrix components (Boles et al. 2008;Zhang et al. 2018).Therefore, the Agr system is a key factor in regulating the formation of S. aureus biofilm.In our study, we found that 4 µg/mL of AKBA can not only inhibit the biofilm formation, but also significantly eradicate the pre-formed biofilm against S. aureus.Therefore, we speculate that the anti-biofilm mechanism of AKBA could be mediated by its interference with the Agr system.However, further study is needed in the future.
AKBA has strong antibacterial activity against MRSA and its clinical strains with MICs of 4-8 µg/mL.AKBA can also inhibit the biofilm formation of MRSA and effectively remove the pre-formed biofilm.AKBA hardly induces the resistance formation of S. aureus, and its

Fig. 4 Fig. 5
Fig. 4 Antibiofilm effects of AKBA.(A) Biofilm inhibition activity of AKBA determined by crystal violet staining.(B) Biofilm eradication activity of AKBA determined by crystal violet staining.(C) Biofilm inhibition visualization by SYTO9/PI staining.(D) Fluorescence intensity quantitation of (C).(E) Biofilm eradication visualization by SYTO9/PI staining.(F) Fluorescence intensity quantitation of (E).The biofilm was stained with SYTO9/PI fluorescent probes.SYTO9 (green) and PI (red) were indicated viable cells and dead cells, respectively.(G) Persister killing activity of AKBA determined by time-killing assay.VAN/CIP/DAP/GEN at the concentration of ten-fold of MIC were used as controls and Staphylococcus epidermidis RP62A and ATCC 12,228 were purchased form American Type Culture Collection or BNCC BeNa Culture Collection (Beijing, China).Other type strains of Escherichia coli ATCC 25,923, Enterococcus faecalis ATCC 29,212, Klebsiella pneumoniae ATCC 700,603, Acinetobacter baumannii ATCC 19,606 and Pseudomonas aureginosa PAO1 were provided by Luo Juncai

Table 1
Antimicrobial susceptibility of AKBA against Grampostive/negative pathogens a: MRSA

Table 2
Synergistic antimicrobial activity between AKBA and GEN against MRSA and MSSA clinical isolates

Table 3
Synergistic antimicrobial activity between AKBA and AMK against MRSA and MSSA clinical isolates